Tissue-selective insulin analogs

ABSTRACT

Human insulin analogs are disclosed. These analogs are tissue-selective. Accordingly, pharmaceutical formulations containing the analogs of the invention provide superior clinical benefits as compared to human insulin when used in the treatment of patients suffering from diabetes. The analogs are modified at amino residue A12, A15 or A19, are different from the naturally occurring residue at said position, and are hepatoselective. Also disclosed are human insulin analogs modified at amino acid residues A12 or A14 or amino acid residues A10 and A13 different from naturally occurring residues or residues at said position or positions and are peripheral selective. DNA sequences and microorganisms comprising sequences coding for human insulin analogs are also provided. Processes for preparing the human insulin analogs are described.

CROSS REFERENCES

This application is a continuation-in-part of our pending U.S.application Ser. No. 07/741,938 filed Aug. 8, 1991, which application isincorporated herein by reference in its entirety and to whichapplication we claim priority under 35 USC §120.

FIELD OF THE INVENTION

The present invention relates generally to biologically active proteinsand analogs thereof. More specifically, the invention relates to analogsof human insulin which analogs are tissue-selective and topharmaceutical formulations containing such analogs and their use in thetreatment of diabetes. DNA sequences and microorganisms comprisingsequences coding for human insulin analogs are also provided. Processesfor preparing the human insulin analogs are described.

BACKGROUND OF THE INVENTION

Insulin is a protein and more specifically a hormone that controls themetabolism of glucose. Lack of insulin within an animal results in theanimal developing diabetes and excess amounts of insulin results incoma. Insulin is a polypeptide which is produced by the beta-cells ofthe islets of Langerhans of the pancreas. Pancreatic secretions ofinsulin are stimulated by high blood levels of glucose and amino acidsafter meals. Glucose uptake is then stimulated by the action of insulinon various tissues (e.g., muscles, liver, and fat). Insulin alsostimulates glycogen and fat synthesis. Pharmaceutical preparations ofinsulin are used therapeutically in the treatment of diabetes mellitusknown as type I and type II diabetes.

The inability of certain animals, such as humans, to generate sufficientamounts of insulin in the pancreas leads to the development of diabetesmellitus. Diabetes mellitus is a syndrome characterized by abnormalinsulin secretion and various metabolic and vascular impairments.Individuals suffering from diabetes mellitus are currently treated bythe administration of porcine, bovine or recombinant human insulin. Theadministration of insulin, however, does not consistently mimic theeffects of endogenous insulin and may result in hypoglycemia andlong-term complications such as atherosclerosis.

Animal derived insulin is not chemically identical to human insulin andsometimes contains other biologically active impurities. Efforts weremade to develop a means of producing insulin which is chemicallyidentical to human insulin and which does not include any biologicallyactive impurities. As a result of such efforts, recombinant techniqueshave been developed to produce human insulin and proinsulin polypeptidesin microorganisms which are isolated and purified for pharmaceuticaluse. For example, see European Patent Application No. 0 055 945,published 14 Jul. 1982, which application is incorporated herein byreference, for its disclosure of recombinant techniques andmethodologies for producing insulin and proinsulin polypeptides and theisolation of such polypeptides.

As indicated above, present technology makes it possible torecombinantly produce insulin having an identical amino acid sequence tohuman proinsulin and/or insulin which can then be purified and used inpharmaceutical preparations. However, insulin has a plurality offunctions. For example, it inhibits hepatic glucose production andstimulates peripheral glucose utilization thereby controlling themetabolism of glucose. Because of these different functions and becausethe site of administration of the exogenous insulin typically isdifferent from the natural release site, when individuals suffering fromdiabetes are treated it may be desirable if the insulin which isadministered inhibited hepatic glucose production to a greater degreecompared with its ability to stimulate peripheral glucose utilization.The need for the insulin administered to act differently from naturalinsulin in order to obtain a more natural result is explained furtherbelow.

Insulin has an overall effect of lowering the glucose concentration inthe bloodstream. This effect is obtained by the operation of insulin ontwo different types of tissue.

The glucose lowering effects of insulin occur in both hepatic andperipheral tissues in order to regulate glucose levels in blood. Theinteraction of insulin with hepatic receptors results in a decrease inglucose production by the liver as well as increased liver storage ofglucose as glycogen; interaction with the peripheral receptors on fatand muscle cells results in an increase in glucose utilization. Bothinteractions result in a lowering of glucose concentration in thebloodstream. In both liver and peripheral cells, binding to the receptoris concomitant with insulin clearance from the system; i.e., as insulinis utilized, it is also cleared.

In the normal operation of endogenous insulin, the majority of thehormone secreted by the pancreas interacts with the hepatic receptors.This apparent preference is thought to be due to the proximity of theliver to the source of the hormone. Once released into the generalcirculation, most of the insulin appears to be utilized by peripheralcells, due to the large number of peripheral receptors available. Onereason why the administration of insulin does not achieve the "natural"balance between hepatic and peripheral activity may be that the initialintroduction of the drug into the general circulation system provideslittle opportunity for interaction with the hepatic receptors. However,a high degree of such interaction takes place when endogenous insulin isreleased from the pancreas.

Insulin is initially synthesized in the islets of Langerhans of thepancreas as the single chain peptide proinsulin. Proinsulin has 1-2% ofthe potency of native insulin when assayed in vitro, about 15% of thepotency of insulin in vivo. and a circulating half-life of 30 min ascompared to 4 min for insulin. Proinsulin is relatively hepatoselectivein vivo (Glauber, H. S., et al., Diabetes (1986) 35:311-317; Peavy, D.E., et al., J. Biol. Chem. (1985) 260:13989-13994; Davis, S. N., et al.,Diabetes (1988) 37:74 (abstract)). The in vivo hepatoselectivity ofproinsulin is 50% more than that of insulin per se. However, proinsulinhas too low a potency for most uses.

Insulin is thought to circulate predominantly as a monomer. The monomeris a disulfide-linked, two-chain molecule consisting of A chain of 21amino acids and B chain of 30. The amino acid sequences of human,porcine and bovine insulin are well established. (The amino acidsequences of insulins of many other species have also been determined.)Attempts to discern which are the essential residues in these peptideswere begun some time ago. By observing the conservation of residuesbetween the insulins derived from various species, it was suggested thata largely invariable region on the surface of the monomer is thereceptor binding region. This region includes A-chain residues Al (Gly),A5 (Gln), A19 (Tyr), and A21 (Asn) as well as B chain residues B24(Phe), B25 (phe), B26 (Tyr), B12 (Val), and B16 (Tyr).

de Meyts R. A. et al. Nature (1978) 273:504-509, tested 29 insulin-typemolecules including animal insulins and proinsulins, insulin-like growthfactors and chemically modified insulins for ability to bind to receptorand for biological potency. de Meyts et al. found a one thousand-foldvariation over the series of 29 analogs wherein the essential residueswere shown to be some of the 8 carboxyterminal residues of the B chainand the A21 (Asn) residue of the A chain.

Tompkins, C. B., et al., Diabetologia (1981) 20:94-101, showed thatcertain analogs stimulated hypoglycemia entirely by increasingperipheral glucose uptake, whereas others did so by decreasing hepaticglucose production. In these studies, A1, B29-diacetyl derivatives ofinsulin were able to stimulate peripheral glucose uptake, while A1-B29cross-linked insulins and proinsulin decreased hepatic glucoseproduction.

Later studies by Nakagawa, S. H., et al., J. Biol. Chem (1986)261:7332-7341, confirmed the importance of the carboxy terminal regionof the B chain. Studies of binding to the hepatocyte receptor showedthat insulin residues B26-B30 could be deleted without decrease inbinding or biological potency when the carboxyl group isalphacarboxamidated to preserve the hydrophobic character of the carboxyterminal B chain domain. However, deletion of residues B25-B30 orB24-B30 resulted in a decrease in potency.

A reduction in potency was also observed when the phenylalanine at B25was replaced by leu or ser or by homophenylalanine; however, replacementby naphthyl-1-alanine or naphthyl-2-alanine at B25 decreased bindingactivity to a lesser extent. The decreased activity effected byreplacement of Phe at B25 by Ala, Ser, Leu or homophenylalanine wasreversible by deletion of the remaining carboxy-terminal residuesB26-B30. The authors concluded that steric hindrance involving thecarboxy-terminal domain of the B chain helped direct interaction ofinsulin with its receptor, that the negative effect of this domain is"reversed by filling of a site reflecting interaction of the receptorand the beta-aromatic ring of B25 (phe)" and that the remainingcarboxy-terminal residues, besides B25, were important in effecting theinteraction of this residue with the receptor. Further studies by thisgroup (Nakagawa, S. H., et al., J. Biol. Chem (1987) 262:1254-1258)showed that the downstream residues must be deleted to reverse theeffect of replacement of B25 (Phe) by ser and that replacement ofresidues B26 (Tyr) or B27 (Thr) does not reverse this decrease inaffinity. It was further shown that cross-linking between B29 (Lys) andA1 (Gly) decreases the affinity of insulin for the receptor. Thesestudies were directed to an effort to enhance the potency of insulin.

Coincidentally, however, a diabetic patient was shown to produce amutant form of insulin having Leu instead of Phe at B25. Two otherpatients were shown to have mutations at the codons for B24 or B25, butthe encoded insulin was not characterized (Shoelson, S., et al., Nature(1983) 302:540-543).

Increased binding to receptor and increased potency was shown to be aproperty of insulin iodinated at the tyrosine residue B26 (Podlecki, D.A., et al., Diabetes (1983) 32:697-704). Similarly, Schwartz, G. P., etal., Proc. Natl. Acad. Sci. USA (1987) 84:6408-6411, described asuperactive insulin with enhanced binding both to hepatic and peripheralreceptors which contains an aspartic acid substitution for the naturalhistidine at B10 of human insulin.

Still others have reported insulin analogs which have specifiedproperties thought desirable. For example, Brange, J., et al., Nature(1988) 333:679-682, prepared analogs with substitutions at B9, B12, B10,B26, B27 and B28 which are designed to prevent formation of dimers.

International Patent Application WO 90/12814, published 1 November 1990,discloses hepatospecific insulin analogs wherein tryptophan or otherbulky, hydrophobic residues selected from the group consisting oftryptophan, naphthylalanine, N-gamma-dansyl-alpha, gamma-diaminobutyricacid, leucine, valine, phenylalanine and other hydrophobic amino acidsare substituted at the A13, A14, A15, A19 and B16 positions of theinsulin polypeptide. The naturally occurring amino acids which areconventionally described as hydrophobic are alanine, valine, leucine,isoleucine, proline, methionine, phenylalanine and tryptophan, oralternatively amino acids whose side chains consist only of hydrocarbon,except for the sulfur atom of methionine and the nitrogen atom oftryptophan (J. Darnell, Molecular Cell Biology, Scientific AmericanBooks, New York, 1986). In contrast, the sidechains of histidine andglutamine are described as polar or hydrophilic groups.

Contrary to the teaching in WO 90/12814, the present invention providesthe surprising and beneficial result that substitution bynon-hydrophobic amino acids at A19 produces insulin analogs which arehepatoselective in vivo. Further, the present invention provides thesurprising and beneficial result that substitution by phenylalanine atA14 produces analogs that are peripheral selective. Such A14 analogshave a different and distinct therapeutic use from the hepatospecificuse disclosed in International Patent Application WO 90/12814.

The present invention recognizes the desirability of tissue selectivitywhen providing insulin to a patient from an exogenous source, i.e., notdirectly from the pancreas.

SUMMARY OF THE INVENTION

Purified human insulin analogs which are tissue selective are disclosed.By tissue selective is meant hepatoselective and peripheral selectiveinsulin analogs. Hepatoselective analogs inhibit hepatic glucoseproduction in vivo to a greater extent than they stimulate peripheralglucose utilization, as compared to normal human insulin activity whengiven by the same route of administration. Peripheral selective analogsinhibit hepatic glucose production in vivo to a lesser extent than theystimulate peripheral glucose utilization, as compared to normal humaninsulin activity when given by the same route of administration.

An object of the invention is to provide human insulin analogscomprising an amino acid residue at position A12 different from thenaturally occurring residue at said position, wherein the analog ishepatoselective. In one embodiment of the invention, the human insulinanalog comprises A12Gly.

Another object of the invention is to provide human insulin analogscomprising an amino acid residue at position A19 different from thenaturally occurring residue at said position and which is not ahydrophobic residue, wherein the analog is hepatoselective. In oneembodiment of the invention, the human insulin analog comprises A19Hisor A19Gln.

Another object of the invention is to provide human insulin analogscomprising an amino acid residue at position A12 different from thenaturally occurring residue at said position, in combination with one orboth of an amino acid residue at A19 different from the naturallyoccurring residue at said position and an amino acid residue at A15different from the naturally occurring residue at said position, whereinthe analog is hepatoselective. In one embodiment of the invention, aglycine residue may be substituted for the naturally occurring residueat the A12 position and a histidine residue may be substituted for thenaturally occurring residue at the A19 position.

Another object of the invention is to provide human insulin analogscomprising an amino acid residue at position A12, or amino acid residuesat position A10 and A13 in combination, wherein said residue or residuesare different from the naturally occurring residue or residues at saidposition or positions and wherein the analog is peripheral selective. Inone embodiment of the invention, an alanine residue may be substitutedfor the naturally occurring residue at the A12 position; or a prolineresidue may be substituted for the naturally occurring residue at theAl? position and a tryptophan may be substituted for the naturallyoccurring residue at the A13 position.

Yet another aspect of the invention is to provide a pharmaceuticallyeffective amount of a peripheral selective human insulin analog whichmay be administered to an animal in need of peripheral selective insulintreatment, wherein the amino acid residue at position A14 isphenylalanine.

Accordingly, such tissue-selective analogs can be included, individuallyor in combination, within pharmaceutical formulations comprising apharmaceutical excipient material and a pharmaceutically effectiveamount of a human insulin analog which can be administered ininjectable, pump, or other forms to an individual in need thereof toeffectively treat type I and type II diabetics.

Yet another object of the invention is to provide DNA sequences andmicroorganisms comprising sequences coding for human insulin analogs.Another object is to provide processes for preparing the human insulinanalogs.

A primary object of the invention is to provide purified, chemicallysynthesized, or recombinant human insulin analogs.

An advantage of the present invention is that the insulin analogs can beformulated into pharmaceutical preparations which can effectively treattype I and type II diabetics.

Another advantage of the present invention is that it provides for atreatment regime for diabetics and other individuals which has superiorclinical benefits as compared to treatment regimes using insulinformulations wherein the insulin is identical in structure to humaninsulin.

These and other objects, advantages, and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis, and usage as more fullyset forth below, reference being made to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and SEQ ID NOS:1-6 show a representation of the six chemicallysynthesized oligonucleotides that are combined in a specific sequenceand comprise a gene that codes for human proinsulin.

FIG. 2a and SEQ ID NOS:7-8 are representations of the DNA sequenceencoding amino acids 1-73 of CAT, an 8 amino acid acid linker sequence,and human proinsulin. Also shown is a representation of the deducedamino acid sequence encoding human proinsulin protein. The location ofthe oligonucleotides described in FIG. 1 used to construct theproinsulin cDNA is given in FIG. 2b SEQ ID Nos.:16-18.

FIG. 3 shows glucose turnover in euglycemic, hyperinsulinemic clamps forinsulin and the A12Gly analog. Graph A shows the rate of exogenousglucose infusion (GIR). Graph B shows the rate of glucose disappearance(R_(d)). Graph C shows the hepatic glucose output and is calculated asR_(a) minus GIR. Under steady state conditions, R_(a) =R_(d).

FIG. 4 shows glucose turnover for insulin, the A19Gln analog and theA19His analog. Graph A shows the rate of exogenous glucose infusion.Graph B shows the rate of glucose disappearance (R_(d)). Graph C showsthe hepatic glucose output (HGO) and is calculated as R_(a) minus GIR.

DETAILED DESCRIPTION OF THE INVENTION

Before the present human insulin analogs, processes for making such andformulations containing and administering such are described, it is tobe understood that this invention is not limited to the particularformulations and processes described, as such formulations andmethodology may, of course, vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms "a," "an" and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "a formulation" includes mixtures of such formulations,reference to "an analog" includes mixtures of analogs, and reference to"the method of recombinant synthesis" includes one or more such methodsof the type described herein, and so forth. The term human insulinanalog, as used in the specification and appended claims, is not to beconstrued as implying that said analogs are necessarily derived directlyor indirectly from a human source.

The present invention provides purified human insulin analogs which aretissue-selective. Hepatoselective insulin analogs have a greateractivity at the liver than at the periphery when compared to theactivity of insulin when given by the same route of administration.Accordingly, such tissue-selective analogs can be placed inpharmaceutical formulations and administered to diabetics to obtainsuperior clinical benefits as compared to human insulin. The amino acidsequence of each of the hepatoselective analogs of the invention isidentical to the amino acid sequence of human insulin except at (a)position A12, wherein the amino acid residue is different from thenaturally occurring residue at said position, or (b) position A19,wherein the amino acid residue is different from the naturally occurringresidue at said position and is not hydrophobic, or (c) position A12 incombination with one or both of position A15 and position A19, whereinthe amino acid residues at said positions are different from thenaturally occurring residues at said positions and wherein the analog ishepatoselective. Additionally, various combinations of the A12, A15, andA19 amino acid substitutions are believed to result in hepatoselectiveinsulin analogs.

The present invention further provides for human insulin analogs whichare peripheral selective. Peripheral selective analogs inhibit hepaticglucose production in vivo to a lesser extent than they stimulateperipheral glucose utilization, as compared to normal human insulinactivity when given by the same route of administration. Individuals whohave a normal fasting glucose but impaired glucose tolerance may takeadvantage of the analogs of the present invention. For such individuals,a peripheral selective insulin analog may be used to stimulateperipheral activity of insulin to a greater extent when compared toactivity of insulin at the liver. Such analogs can be incorporated intopharmaceutical formulations and administered to individuals to obtainsuperior clinical benefits as compared to human insulin. The amino acidsequence of each of the peripheral selective analogs of the invention isidentical to the amino acid sequence of human insulin except at (a)position A12, wherein the amino acid residue is different from thenaturally occurring residue at said position or (b) position A10,wherein the amino acid residue is different from the naturally occurringresidue at said position and at position A13, wherein the amino acidresidue is different from the naturally occurring residue at saidposition. The present invention may also be used to treat an animal inneed of peripheral selective insulin treatment with a pharmaceuticallyeffective amount of a human insulin analog, wherein the amino acidresidue at A14 differs from the naturally occurring amino acid and isphenylalanine.

Any known method may be used to prepare insulin. For example, the cDNAcoding for proinsulin can be constructed using oligonucleotides.Alternatively, the cDNA coding for proinsulin can be isolated from ahuman insulinoma cDNA library using an oligonucleotide screening probe.Isolation of a clone containing the sequence for proinsulin has beendescribed previously by Bell, G. I., et al., Nature (1979) 282:525-527and by Chan, S. J., et al., Proc. Natl. Acad. Sci. (1981) 78:5401-5405.

Both chemical synthesis and recombinant methods may be used to preparethe analogs of the invention. For recombinant material, it may benecessary to synthesize cDNAs, which is done by means known to thoseskilled in the art, such as, for example, by using PCR technology. Whenthe appropriate cDNAs are constructed (e.g., by using DNA which encodesfor proinsulin, making appropriate codon substitution and carrying outPCR), they are operably fused into expression plasmids, which plasmidsare then used to transfect microorganism host cells such as E. colibacteria. The transfected hosts are then allowed to grow and theconstructed cDNAs will express the appropriate proinsulin analogs.

The expressed protein is subjected to standard isolation andpurification procedures in order to obtain substantially purepolypeptides. The proinsulin analogs are preferably expressed as aCAT-analog fusion protein. Accordingly, after the transfected hosts areallowed to express the fusion protein, the fusion protein is isolatedand then subjected to CNBr cleavage in order to separate the CAT fromthe desired proinsulin analog. The proinsulin analog undergoessulfitolysis, is refolded, and undergoes enzymatic cleavage to producethe insulin analog. The insulin analog is then isolated and purifiedusing protein purification procedures known to those skilled in the art.The resulting analogs are refolded polypeptides having the correctdisulfide bonds so that the resulting structure has the desiredbiological activity. The analogs are purified to 95% or purer,preferably 99% or purer for use in patients.

Analogs of the invention can be tested in a euglycemic, hyperinsulinemicrat clamp using ³ H-glucose as a tracer. In accordance with thistechnique, glucose is infused to maintain euglycemia. Insulin or ananalog is also infused. Using ³ H-glucose, glucose utilization by theperiphery (R_(d)) and glucose production by the liver (HGO) can becalculated under steady state conditions using the equation of Steel etal., Am. J. Physiol. (1956) 187:15-24, incorporated herein by reference.

The purified analogs are formulated in injectable pharmaceuticalformulations and administered in order to treat type I and type IIdiabetes. The amount of an insulin analog to be administered in apharmaceutical composition is typically in the range of about 0.1 mg toabout 10 mg per day.

Insulin analogs, for example, can be formulated into pharmaceuticalcompositions suitable for parenteral or nasal administration withappropriate excipients or carriers. Any of a variety of techniques knownin the art can be used to prepare the pharmaceutical compositions. See,for example, Remington's Pharmaceutical Sciences. 17th Edition, MackPublishing Company, Easton, Pa. (1985), which is incorporated herein byreference.

For example, the human insulin analogs described herein may beformulated in such a manner as to allow for their delivery by a varietyof routes of administration, including, but not limited to intravenousinjection, subcutaneous injection, and intramuscular injection. It iswell recognized that insulins (e.g., human, bovine and porcine insulins)may be prepared in various formulations for subcutaneous orintramuscular injection, which, depending on the pH, zinc content,preservative, isotonic agent, buffer, and added basic proteins such asprotamine, provide varied degrees of absorption when injectedsubcutaneously. See for example, Jens Brange, Galenics of Insulin, ThePhysico-chemical Aspects of Insulin and Insulin Preparations,Springer-Verlag, New York, 1987. Two general classes of formulation areparticularly useful, and are known variably as "soluble, regular orrapid-acting" and protracted-acting derivatives. Such protracted-actingformulations fall into two subclasses: (1) "isophane," "NPH" orintermediate-acting and (2) "lente," "long-acting." Soluble formulationsmay be prepared in either acid or neutral form, but the neutral form maybe better tolerated at the injection site, and also may reducedecomposition of the insulin analog on storage.

Crystalline zinc insulin analogs, suitable for use in pharmaceuticalpreparations may be prepared from a mixture containing approximately1.5% of the insulin analog, 7% sodium chloride, 0.1 molar sodiumacetate, and a quantity of zinc ions (from zinc chloride or zincacetate) adequate to give a total of 0.8-0.9% of zinc by weight of theinsulin analog, corresponding to approximately 4 zinc atoms per sixmolecules of the insulin analog. Adjustment of the pH of the solution byaddition of a mineral acid, such as aqueous hydrochloric acid, to pH 5.5or the isoelectric point of the analog if different from native insulin,causes crystals or an amorphous precipitate to form. This may beaccelerated by addition of previously formed insulin analog crystals. onstanding, the precipitate may gradually redissolve, and crystallinematerial forms, until a new equilibrium state is reached in which thecrystalline form predominates. It is well known to those skilled in theart that addition of chemical compounds that interact either with zincions or the insulin molecules will change the crystallization pattern,and the optimal conditions for crystallization may change somewhatdepending on the specific insulin analog. The crystalline form of theinsulin analog so obtained may also be altered by addition of additivessuch as phenol or other hydroxylated aromatic compounds, as well as bysubstantial changes in the zinc content or the pH of the medium.

The crystalline insulin analogs may be used to prepare variousformulations of the insulin analogs, which, depending on a number offactors, including added cationic organic compounds, such as globin,added basic proteins such as protamine, added zinc ion, or size orphysical state of the insulin analog particles or crystals may havevaried absorption rates from the site of injection, and hence variedonset and duration of action.

For example, a soluble, rapid acting form of the insulin analog forsubcutaneous, intramuscular or intravenous injection may be prepared bydissolving the zinc insulin analog crystals in dilute hydrochloric acidat pH 3 to an insulin analog concentration of approximately 4%. Aftersterilization by filtration, additives for preservation of sterility,such as phenol (0.2%) or methylparaben (0.1%), isotonic agent (such assodium chloride (0.7%) or glycerol (1.6%), buffering agent (such assodium acetate 0.01M) may be added, such that their concentrations inthe final formulation are approximately those shown in parentheses. ThepH of the solution is adjusted carefully to neutrality (pH 7) withdilute alkali, such as 0.01 to 0.1N sodium hydroxide, in such a manneras to avoid attainment of locally high pH and degradation of the insulinanalog. The neutral solution is then diluted with sterile water to thedesired final insulin analog concentration, usually between 1 mg/mL and10 mg/mL, and desired ionic strength, preferably isotonic with humanserum, by addition of sterile water. Such a formulation may requirestorage at sub-ambient temperature, above 0° C., preferably at 4° C. toreduce degradation before use.

More protracted preparations, known as isophane formulations, may beprepared by addition of basic proteins, such as protamine (e.g.,salmine), to a solution of insulin analog at neutral pH in equal(isophane) proportions. In the presence of added zinc ions approximately0.2 μg of zinc ion per 25 mg of insulin analog), and a phenoliccompound, either phenol or a cresol, a complex of the protamine and theinsulin analog is formed as a precipitate, which has a more delayedabsorption from the injection site than soluble insulin analog. It iswell known to those skilled in the art, enabled by the disclosureherein, that the nature of the preparation so obtained, particularly theratio of protamine and insulin analog in the precipitate, may beaffected by modification of the pH, temperature, concentration of addedzinc, and auxiliary substances. These variables may affect theabsorption of insulin analog and hence onset and duration of action ofthe resulting formulation following subcutaneous or intramuscularinjection.

An even more protracted-acting, or utralente formulation, may beobtained by increasing the amount of zinc present in the formulation.Such formulations may be prepared by adding zinc ions, from either zincacetate or zinc chloride, to a suspension of crystalline insulin analog,such as that described in the preceding description for the preparationof insulin analog crystals, with the exception that phenol should beavoided. A final concentration of total zinc ion of approximately 0.09to 0.15 mg/mL is desirable, depending on the final concentration ofinsulin analog in the formulation, the optimal amount being that whichallows a concentration of approximately 0.05 mg/mL of free zinc ion insolution (not complexed to the insulin analog precipitate). It is knownto those skilled in the art, enabled by the disclosure herein, that thesize and variability of the crystals obtained in this manner may affectaction of the resulting formulation following subcutaneous orintramuscular injection.

Another protracted form, or lente formulation, may be prepared bydissolving the free insulin analog at acid pH (e.g., pH 3 in dilutehydrochloric acid), such that the insulin analog concentration is in therange of 4% and, after sterile filtration, zinc is added (as zincchloride or zinc acetate) such that a final zinc concentration of 0.09to 0.15 mg/ml is obtained. The pH is then adjusted carefully toneutrality with dilute alkalai such that an amorphous precipitate forms.It is well known to those skilled in the art, enabled by the disclosureherein, that the nature of the precipitate so obtained may vary with thefactors influencing the precipitation, such as rate of neutralization,or concentration of the insulin analog or zinc or other additives, andthis may affect the absorption of the insulin analog and hence rate ofonset and duration of the resulting formulation following subcutaneousor intramuscular injection.

It will be apparent to those skilled in the art, enabled by thedisclosure herein, that other formulations of the insulin analogsdescribed herein may be obtained by modification of the above generalprocedures, leading to a broad range of formulations with varied ratesof absorption following subcutaneous or intramuscular injection, andhence varied onsets and durations of action. It is also well known tothose skilled in the art, that insulin molecules may be formulated in amanner such as to allow for delivery by non-parenteral routes, such asby oral, nasal, rectal, and conjunctival routes. The insulin moleculesdescribed in the application should also be suitably delivered informulations where native insulins (e.g., human, bovine or porcine) havebeen delivered by these routes.

It should be understood that the effective amount of all or any of theinsulin analogs administered to patients will be determined by thecaregiver and will be based on criteria such as the size, age and sex ofthe patient, route of administration, as well as patient responsivenessand the degree to which the patient's pancreas fails to produce insulin.Patients can be started on dosing regimes similar to those which thepatient was accustomed to with respect to insulin. Thereafter, theamount of the tissue-selective insulin analogs administered can beadjusted based on patient responsiveness.

EXPRESSION OF THE INSULIN FUSION PROTEIN

Insulin analogs are provided as fusion proteins expressed in bacterialcells. It is well known that many eucaryotic proteins are incapable ofbeing expressed in bacterial cells in measurable amounts and areincapable of being expressed at commercially recoverable levels due toproteolysis of the foreign protein by the host. As described incopending, commonly assigned application Ser. No. 07/391,277, filed 8August 1989 (published through the PCT as WO 90/01540 on 22 February1990) and incorporated herein by reference, proteins which cannot beexpressed in high yield may be expressed as a fusion protein to increaselevels of expression. For purposes of the present invention, it ispreferred that the insulin analogs be expressed as fusion protein withchloramphenicol acetyltransferase (CAT), which is a known selectablemarker and an easily assayed enzyme for monitoring the efficiency ofboth eucaryotic and procaryotic expression (Delegeane, A. M., et al.,Molecular Cell Biology (1987) 7:3994-4002). The fusion proteins whichone begins with in the isolation and purification methods of the presentinvention are substantially of the same general type as described in theaforementioned patent application. The following is a brief summary ofthe fusion and expression process set forth in the aforementioned patentreference.

CAT encodes a 219 amino acid mature protein and the gene contains anumber of convenient restriction endonuclease sites (5'-PvuII, EcoRI,DdeI, NcoI, and ScaI-3') throughout its length to test gene fusions forhigh level expression. These restriction sites may be used inconstructing hybrid gene sequences.

Expression constructs using CAT can employ most of the CAT-encoding genesequences or a substantially truncated portion of the sequence encodingan N-terminal portion of the CAT protein linked to the gene encoding thedesired heterologous polypeptide. These expression constructs, whichdemonstrate enhanced levels of expression for a variety of heterologousproteins, utilize a number of varying lengths of the CAT proteinsranging in size from 73 to 210 amino acids. The 73 amino acid CAT fusioncomponent is conveniently formed by digesting the CAT nucleotidesequence at the EcoRI restriction site. Similarly, the 210 amino acidCAT fusion component is formed by digesting the CAT nucleotide sequencewith ScaI. These, as well as other CAT restriction fragments, may thenbe ligated to any nucleotide sequence encoding a desired protein toenhance expression of the desired protein.

The reading frame for translating the nucleotide sequence into a proteinbegins with a portion of the amino terminus of CAT, the length of whichvaries, continuing in-frame with or without a linker sequence into theproinsulin analog sequence, and terminating at the carboxy terminusthereof. An enzymatic or chemical cleavage site may be introduceddownstream of the CAT sequence to permit ultimate recovery of thecleaved product from the hybrid protein. Suitable cleavage sequences andpreferred cleavage conditions will be described below.

To avoid internal cleavage within the CAT sequence, amino acidsubstitutions can be made using conventional site-specific mutagenesistechniques (Zoller, M. J., and Smith, M., Nucl. Acids Res. (1982)10:6487-6500, and Adelman, J. P., et al., DNA (1983) 2:183-193). This isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. These substitutionswould only be performed when expression of CAT is not significantlyaffected. Where there are internal cysteine residues, these may bereplaced to help reduce multimerization through disulfide bridges.

Procaryotic systems may be used to express the CAT fusion sequence;procaryotes most frequently are represented by various strains of E.coli (e.g., MC1061, DH1, RR1, W3110, MM294, MM294B, C600hf1, K803,HB101, JA221, and JM101), however, other microbial strains may also beused. Plasmid vectors which contain replication sites, selectablemarkers and control sequences derived from a species compatible with thehost are used, for example, E. coli is typically transformed usingderivatives of pBR322, a plasmid derived from an E. coli species byBolivar et al., Gene (1977) 2:95. pBR322 contains genes for ampicillinand tetracycline resistance and thus provides multiple selectablemarkers which can be either retained or destroyed in construction of thedesired vector.

In addition to the modifications described above which would facilitatecleavage and purification of the product polypeptide, the geneconferring tetracycline resistance may be restored to exemplified CATfusion vectors for an alternative method of plasmid selection andmaintenance.

Although the E. coli tryptophan promoter-operator sequences arepreferred, different control sequences can be substituted for the trpregulatory sequences. Commonly used procaryotic control sequences whichare defined herein to include promoters for transcription initiation,optionally with an operator, along with ribosome binding site sequence,include such commonly used promoters as the beta-lactamase(penicillinase) and lactose (lac) promoter systems (Chang et al., Nature(1977) 198:1056), the lambda-derived P_(L) promoter (Shimatake et al.,Nature (1981) 292:128) and N-gene ribosome binding site, and the trp-lac(trc) promoter system (Amann and Brosius, Gene (1985) 40:183).

Transformed microorganisms producing the fusion proteins are grown in asuitable growth medium containing compounds which fulfill thenutritional requirements of the microorganism. Growth media willtypically contain assimilable sources of carbon and nitrogen, magnesium,potassium and sodium ions, and optionally, amino acids, purine andpyrimidine bases, vitamins, minerals, and the like.

At the end of fermentation, the bacterial paste is collected by, e.g.,cross-flow filtration, centrifugation, or other conventional methods.The concentrated paste is preferably stored at a temperature below -20°C. preferably about -70° C., until further use.

Cell Disruption and Preparation of Inclusion Bodies

Following concentration of the bacterial paste, the cell membranes andcell walls of the microorganisms are disrupted, either chemically, i.e.,with alkali or with a compound such as 1-octanol, enzymatically, e.g.,with lysozyme, or mechanically, i.e., using a commercially availablehomogenizer or microfluidizer, as is well-known in the art. The endpoint of disruption can be monitored by microscopy and/or by adding adye such as Coomassie Blue and monitoring its absorbance at 595 nm,which typically increases with cell lysis. This process step should becarried out for a time long enough to ensure that substantially all ofthe cells have been disrupted, and that substantially no intact cellswill be carried through to subsequent process steps.

After cell disruption, the insoluble fraction of the whole-cellhomogenate, containing inclusion bodies, is harvested by filtration,centrifugation, or the like. The inclusion body fraction is typically onthe order of 10 to 30% of the initial wet-cell weight, and enrichmentfor the CAT/insulin analog fusion protein is thus desirable. To removecontaminating bacterial proteins, the inclusion body pellets are washedwith a medium which contains 1 M guanidine hydrochloride together withdithiothreitol (DTT), e hylene diamine tetracetic acid (EDTA), andbuffering agents. After washing, the inclusion bodies are pelleted bycentrifugation and washed again with the aforementioned medium. Thiswashing procedure is important to achieve relatively high (50-70%)yields of insulin analog. By washing away part of the contaminatingbacterial protein, this procedure increases the proportion of inclusionbody protein that is CAT-insulin analog fusion protein to about 50%. Theinclusion bodies may, if desired, be frozen and stored.

CLEAVAGE OF THE FUSION PROTEIN

The reagent and methods used in cleaving the fusion proteins will dependon the cleavage sequence incorporated in the fusion protein at theoutset. Cleavage sequences which may be used herein include, forexample, those which cleave following methionine residues (cleavagereagent cyanogen bromide), glutamic acid residues (cleavage reagentendoproteinase Glu-C), tryptophan residues (cleavage reagentN-chlorosuccinimide with urea or with sodium dodecyl sulfate), andcleavage between asparagine and glycine residues (cleavage reagenthydroxylamine). For purposes of the present invention, cleavage withcyanogen bromide is particularly preferred.

The inclusion bodies obtained in the previous step, in solubilized form,are treated with the selected cleavage reagent at ambient temperature.The reaction is allowed to proceed for as long as necessary to ensuresubstantially complete cleavage of the fusion protein.

After the cleavage reaction is complete, the sample is dried. The driedcleavage mixture is subjected to sulfitolysis in order to modify thefree sulfhydryl groups with S-sulfonates. The modified proinsulin analogin its stabilized S-sulfonate form is partially purified and can berefolded and converted to the active human insulin analog by knownprocedures. Refolding techniques allow the formation of the S--S bonds.The analog is folded to obtain the formation of disulfide bridges andconverted to insulin. Finally, the insulin analog is purified by highpressure liquid chromatography (HPLC).

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the claimedinvention. Specifics are given for construction of A12Gly insulinanalogs, however, the procedure can be used to prepare other insulinanalogs of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake, formulate and administer the insulin analogs and insulin analogpharmaceutical formulations of the invention, and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to insure accuracy with respect to numbers used (e.g.,amounts, temperatures, etc.), but some experimental errors anddeviations should be allowed for. Unless indicated otherwise, parts areparts by weight, temperatures are in degrees centigrade, and pressure isat or near atmospheric.

Construction of Insulin Analogs

Insulin analogs are constructed my making nucleotide changes in theproinsulin cDNA molecule such that the resulting cDNA molecule encodesthe desired amino acid substitution in the analog.

Proinsulin cDNA is constructed by combining a series of chemicallysynthesized oligonucleotides comprising a gene coding for humanproinsulin and a linker sequence at the 5' end. The oligonucleotides,numbered 1-6 are described in FIG. 1 (SEQ ID NOS:1-6). The analogs arepaired 1 with 2, 3 with 4, and 5 with 6. Each pair is phosphorylated,annealed and then ligated as follows. For the kinase reaction, 5 μg eachof an oligonucleotide pair is brought to 100 μl in 70 mM Tris pH 7.6, 10mM MgCl:, 5 mM DTT, 1 mM ATP and 20 units T4 polynucleotide kinase isadded. The mixture is incubated at 37° C. for 1 hour and stopped byheating at 65° C. for 10 minutes. Oligonucleotides are annealed byheating the above samples to 100° C. for 3 minutes followed by a 30minute incubation at room temperature and, then, incubating overnight at4° C.

The pairs of annealed oligonucleotides are mixed (pooled and ligated byadding 10 mM ATP to bring the concentration to 1 mM in the presence ofT4 DNA ligase. The samples are then incubated at 15° C. overnight. Theligated DNA is then digested with KpnI and HindIII and an approximately280 bp fragment is gel purified by standard techniques.

The cDNA coding for proinsulin is inserted into plasmid vector pUC19(Yannish-Perron, C., et al., Gene (1985) 33:103-119; Roberts, R. J.,Nucl. Acids Res. (1987) 15 Suppl: r189-r217) is digested With KpnI andHindIII by standard techniques. The resulting vector is designatedpPINS. The vector containing the proinsulin DNA (pPINS) is digested withEcoRI and NdeI and ligated with a DNA fragment coding for amino acids1-73 of CAT that is digested with NdeI and EcoRI. The resulting plasmidwill contain DNA sequences which code for amino acids 1-73 of CAT, an 8amino acid linker sequence (FIG. 2a), and human proinsulin. Thisplasmid, pUC-CAT-proinsulin, is used to construct cDNAs for insulinanalogs.

Insulin analogs were constructed using a repetitive series ofenzymatically catalyzed polymerization reactions using an automatedthermal cycler (Perkin Elmer Cetus DNA Thermal Cycler). The constructionof A12Gly is illustrated below. Two oligonucleotide primers wereutilized. One primer was complementary to the antisense strand at theend of the cDNA and has the following sequence (SEQ ID NO:9): 5'-CCG GAATTC GAG CTC GGT ACC CGG-3'. The other primer was complementary to thesense strand at the 3' end of the cDNA except for the nucleotide codonwhich codes for the desired amino acid change. To construct the A12Glyanalog, the oligonucleotide primer complementary to the sense strand,5'-GCC AAG CTT CTA GTT GCA GTA GTT CTC CAG CTG GTA GAG ACC GCA-3' (SEQID NO:10), was used. This primer codes for amino acid Gly at positionnumber 12 rather than amino acid Ser found in proinsulin. A19His isconstructed in the same manner except that the primer complementary tothe sense strand contains the sequence 5'-GCC AAG CTT CTA GTT GCA ATGGTT TTC-3' (SEQ ID NO:11) and codes for amino acid His at position 19rather than the amino acid Tyr found in proinsulin. By using polymerasechain reaction technology (PCR), the desired DNA sequence coding for theamino acid (Gly) is substituted for the naturally occurring amino acid(Ser) at this position. The DNA sequence coding for proinsulin is shownin FIG. 2 and SEQ ID NO:7. For the polymerase chain reaction, themixture was as follows: 10 μl 10×PCR buffer (500 mM KCl, 100 mM Tris-HClpH8.3), 16 μl 1.25 mM deoxynucleotide mix (dATP, dGTP, dTTP, dCTP), 1 μMoligonucleotide primers, 1 ng template pUC-CAT-proinsulin, 10 μl 100%DMSO, 0.5 μl (2.5 units) Taq polymerase, and H₂ O to a final volume of100 μl. The reaction mix was overlaid with a few drops of mineral oiland then the cycler was programmed to repeat the following cycle:

1. Denature at 94° C. for 1 minute;

2. Anneal at 55° C. for 30 seconds;

3. DNA synthesis at 72° C. for 30 seconds.

The amplification reaction was carried out for 30 cycles.

The PCR reaction mixture was then extracted once withphenol:chloroform:isoamyl alcohol (25:24:1) and once withchloroform:isoamyl alcohol (24:1). Sodium acetate (1/10 volume of 3M)and 2 volumes of 100% ethanol were added and the sample incubated for aminimum of 15 minutes at -70° C. The precipitated DNA was collected bycentrifugation in a microfuge for 15 minutes at 4° C. The dry pellet wasresuspended in 45 μl H₂ O and digested with KpnI and HindIII by adding 5μl 10×KpnI buffer (100 mM NaCl, 100 mM Tris-Cl pH 7.5, 100 mM MgCl₂, 10mM DTT), 40 units KpnI, and 40 units HindIII. The approximately 280 bpDNA fragment was purified by electrophoresis on an agarose gel andextracted from the gel. The purified fragment was then inserted byligation into the expression vector pTrp233 (U.S. Pat. No. 4,764,504)containing CAT. The expression vector was digested with KpnI andHindIII. For the ligation mixture, 2.5 μl 10×ligase buffer (0.5 MTris-Cl pH 7.4, 0-1M MgCl₂, 0.1 M DTT), 2.5 μl 10 mM ATP, 20 ng of thedigested expression vector, 2.5 ng PCR DNA fragment, and 1 μl T4 DNAligase (NE Biolabs) was mixed with water to a final volume of 25 μl.

The ligation reaction was allowed to proceed for 2 hours at roomtemperature and then used to transform competent E. coli cells bystandard techniques as described in detail below. From the transformedcells, a single colony was isolated and used to prepare plasmid. Thisplasmid was subjected to DNA sequencing by standard techniques to ensurethat the desired cDNA coding for the insulin analog A12Gly (SEQ IDNOS:12 and 13) was used to transform the cells to be used as a sourcefor the production of recombinant insulin analog.

Production, Isolation and Purification of Recombinant Insulin Analogs

Expression

The human insulin analogs A12Gly (SEQ ID NOS:12 and 13) and A19His (SEQID NOS:14 and 15) were expressed as fusion proteins with portions of thebacterial CAT protein in E. coli. These methods are routinely used topurify insulin analogs expressed as a CAT fusion protein in E. coli. Forpurposes of this example, expression of insulin analogs A12Gly or A19Hiswill be described.

The insulin analog described above was joined to the carboxy terminus ofthe CAT sequence through a cyanogen bromide susceptible methioninelinkage. The CAT-insulin analog fusion was expressed from the tryptophanpromoter of the bacterial expression vector pTrp233.

The plasmid was used to transform E. coli W3110 (ATCC Accession No.27325) and selected for ampicillin resistance. Transformants were grownin culture overnight at 37° C. in complete M9 medium containing M9salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose, 0.5% casmaino acids, 40μg/ml tryptophan, 2 μg/ml thiamine hydrochloride, and 100 μg/mlampicillin sulfate. The overniqht culture (45-50 ml) was used toinoculate 2 L of M9 medium containing 4-8 μg/ml tryptophan. Fusionprotein expression was induced using 25 μg/ml indole acrylic acid bystandard methods when the cells reached an OD₅₉₀ of 0.4-0.5. Cells wereallowed to continue to grow for 4-5 hours at 37° C. Bacterial paste wascollected by centrifugation and stored at -20° C. until use.

Preparation of Inclusion Bodies

A model W-225R sonicator (Heat Systems Ultrasonics, Inc., Plainview,N.Y.) was used to disrupt about 8 g of cells. Cells were suspended in 45ml TE buffer (10 MM Tris pH 7.5, 1 mM EDTA), 1 mM DTT, on ice. Thesonicator was set at 7, constant duty cycle. The blunt tip was used for2 times, 2 minutes with a one minute cooling interval. Samples ofhomogenate were monitored for cell lysis by microscopy. Ten ml of 6 Mguanidine-HCl solution was added to a final concentration of 1 M in 60ml to improve washing of the inclusion bodies. Up to 200 ml of buffer(TE/DTT/guanidine-HCl) were added to further improve the wash. Washedinclusion bodies were pelleted by centrifugation at 5000-7000 rpm for 10minutes in a RC-B GSA rotor, and resuspended in 20 ml ofTE/DTT/guanidine-HCl. The undissolved fraction of the whole cellhomogenate, containing inclusion bodies, was harvested by centrifugationat 5000 X rpm in an SS-34 rotor for 15 minutes. The pellet, containingCAT proinsulin analog fusion protein was stored at -20° C. or subjectedto cyanogen bromide (CNBr) cleavage.

CNBr Cleavage of Fusion Protein

CAT73/proinsulin analog fusion protein was cleaved with CNBr. Inclusionbodies (about 2-3 g) were resuspended in 3 ml of deionized water andthen added dropwise to 16 ml of 88% formic acid. CNBr (120 mg) was addedto solubilized inclusion bodies, the sample was sealed under argon ornitrogen, and then gently stirred at room temperature for 4 hours. Thesample was allowed to dry overnight and the dried residue was stored at-20° C. in a desiccator.

Sulfitolysis

The dried cleavage mixture was redissolved in 12 ml of sulfitolysisbuffer (6 M guanidine-HCI, 50 mM ammonium bicarbonate) and the pHadjusted to 9.0 with ethanolamine (100-200 μl). Two hundred milligramsof sodium sulfite and 100 mg of sodium tetrathionate were added whilestirring, and the pH was readjusted to 9.0 with ethanolamine. Thereaction mixture was stirred at room temperature for 6 hours to convertcysteines and cystines to s-sulfonate groups. The reaction wasterminated by addition of 1/20 volume of 1 M HEPES and about 0.4 ml of 2N HCl to pH 7 and stored overnight at -2° C.

The reaction mixture was desalted on a Sephadex G-15 column in 7 M urea,20 mM Tris pH 7.5. The desalted protein was loaded on a DEAE SepharoseFast Flow column and washed with 7 M urea, 20 mM Tris pH 7.5 followed by50 mM NaCl, 7 m urea, 20 mM Tris pH 7.5. The column was eluted with a200 ml linear gradient of 50-250 mM NaCl using a GM-3 gradient maker(Pharmacia). The proinsulin-analog sulfonate fractions were identifiedby UV trace at 226 nm and/or migration on SDS-PAGE.

Refolding

DEAE fraction pool containing the proinsulin-analog sulfonate wasdesalted on a G-15 column, equilibrated with 50 mM glycine buffer, pH10, and the appropriate fractions, identified by absorbance at 226 nmand were pooled. The Pierce BCA Protein Assay (Pierce, Rockford, Ill.)using Bicinchoninic Acid A was run on the glycine desalted pool ofproinsulin analog sulfonate.

The proinsulin analogs were induced to fold with the correct formationof intramolecular disulfide bonds by controlled sulfhydryl interchangecatalyzed by β-mercaptoethanol. Proinsulin analog glycine pool fractionswere diluted with degassed glycine buffer, pH 10 to a concentration of0.1-0.4 mg/ml and then 0.01 volume of 60 mM β-mercaptoethanol reagentwas slowly added. The sample was blanket sealed under argon, incubatedat 4° C., overnight, and protected from the light.

Enzymatic Transformation

The sample was transformed enzymatically, generally as described inEuropean Patent Application No. 0 264 250, which is incorporated hereinby reference. The refolded sample was concentrated to approximately 2mg/ml. To the proinsulin-analog concentrate in 50 mm glycine buffer pH10 was added 1 M HEPES (to a final concentration of 10 mM), 2 N HCl (toadjust the refold solution to pH 7.2-7.5), 200 mM CaCl₂ (finalconcentration 2.0 mM), and 10 mM NiCl₂ (to a final concentration of0.1mM). Carboxypeptidase B (Worthington, 186 units/mg) at 0.4 mg/ml in10 mM HEPES, pH 7.5 was added to a final concentration of 4 μg/ml.Trypsin-TPCK (Worthington, 225 units/mg) at 0.1 mg/ml, in 10 mM HEPES pH7.5) was added (final concentration 1 μg/ml). The sample was incubatedat 5° C. to -12° C. Conversion was monitored by reversed phase HPLC andcompleted within 5 to 17 hours. Enzymatic transformation was terminatedby addition of 2.5 M acetic acid/ammonium acetate pH 3.5 to a finalconcentration of 0.25 M and stored at 5° C. Acetonitrile (10% v/v) wasadded to samples to be analyzed by HPLC and to inhibit microbial growth.

Analysis of Refold and Enzymatic Transformation

Refolded samples were analyzed after concentration to determine ifproper folding had occurred. Samples (100 μl) for analysis were preparedby dilution with 100 μl of 0.5 m acetic acid and 200 μl of equilibrationmobile phase (20% acetonitrile/0.25 M acetic acid/ammonium acetate pH3.5, followed by mixing and centrifugation to clarify. A 200-400 μlsample was loaded through a loop/injector onto a Toso-Haas TSK ODS 120T5um analytical column, 250×4.6 mm with direct connect guard column andpacked with Vydac pellicular media (Vydac, Hesperia, Calif.).Equilibration was with 20% acetonitrile in 0.25 M acetic acid/ammoniumacetate pH 3.5. Samples were eluted with a linear gradient of 25-40%acetonitrile at 0.5% per minute. The UV absorbance of the columneffluent was monitored at 276 nm. This chromatography system was alsoused to examine the sample after enzymatic transformation. In thisanalysis, the enzymatically transformed species ran earlier than theproinsulin-analog demonstrating the conversion of the proinsulin analogto the insulin analog. This analysis also provided a basis for designingthe preparative HPLC purification of the insulin analog.

HPLC Purification of the Transformed Analog

After enzymatic digestion, the clarified solution was purified on a C18reversed phase high pressure liquid chromatography column (RP-HPLC). Thecolumn was a TSK ODS 120T 10 μ300×7.8 mm column. Up to 30 mg of totalprotein was loaded per run by multiple injections through a 5 ml loop.After the sample was loaded, the column was equilibrated with 0.25 Macetic acid/ammonium acetate pH 3.5/20% acetonitrile and then elutedwith an acetonitrile gradient from 20% to 50%. The insulin analogusually eluted between 28% to 30% acetonitrile. The appropriate peak wasidentified by absorbance at 276 nm and collected. Purified analogfractions were pooled and protein concentration determined. The pooledsample was quick frozen and lyophilized.

For identification and verification of each purified insulin analog,1-20 μg samples were analyzed for peptide sequence and amino acidcomposition. A 5-10 μg sample was analyzed by HPLC to assess purity. AVydac 218TP5415 column was equilibrated in 90% Buffer A/10% Buffer B.(Buffer A is 0.075% TFA (trifluoroacetic acid) in water; Buffer B is0.05% TFA/100% acetonitrile) A linear gradient from 10% to 40% buffer Bover 30 minutes was run at 1.0 ml/min., and the column effluentmonitored at 215 nm. The samples were approximately 85-95% pure.

In Vivo Methods

The in vivo studies were designed to assess the potency of insulinanalogs and to determine if analogs have a tissue selectivity differentfrom insulin.

Analogs were tested using the euglycemic, hyperinsulinemic clamp method.In this study, different rates of insulin analog were infusedconcurrently with variable rates of glucose. The exogenous glucose wasinfused at a rate sufficient to maintain euglycemia for the duration ofthe experiment. Additionally, [D-3-³ H]glucose (tracer) is infused toassess glucose metabolism (i.e, glucose turnover). The steady stateequation of Steele et al. (Am. J. Physiol. (1956) 187:15-241) Was usedto calculate hepatic glucose output and the rate of glucose disposal.Under steady state conditions, glucose (mg/ml) and ³ H-glucose (dpm/ml)levels are constant. Under these conditions the rate of glucoseappearance (R_(a))=rate of glucose disappearance (R_(d)). At steadystate R_(a) =R_(d). R_(a) is further defined as the rate of glucoseentering the glucose pool (approximately the blood volume and otherextra-cellular volume). R_(a) is equal to the rate at which the liver ismaking glucose plus the rate of any exogenous glucose infusion. R_(d) isequal to the rate of glucose leaving the pool.

Three main parameters are used to assess the effects of the analogs. Therate of exogenous infusion (i.e., glucose infusion rate, GIR or M)estimates the potency of analog. R_(d) indicates the degree, relative toinsulin, to which an analog acts at peripheral tissues. Finally, hepaticglucose output (HGO), reflects the ability of the analogs to suppressthe liver's production of glucose. HGO is calculated as R_(a) minus M.To be hepatoselective, the decrease in HGO will be greater than thestimulation of R_(d) compared to that observed with insulin.

Four to five days prior to experimentation, 65 mg/kg of sodiumpentobarbital was administered intraperitoneally to anesthetize rats.Catheters were inserted in the right jugular vein and left carotidartery. The catheters were routed subcutaneously to the dorsal region ofthe neck and exteriorized. Following the recovery period, insulin orinsulin analog plus glucose tracer were infused into the carotid artery.Blood samples were taken from the jugular vein. After collecting basalsample, as described below, insulin or analog infusion was started. At10 minute intervals after starting hormone infusion, 50 μl blood sampleswere taken to determine the plasma glucose level. During infusion ofhormone, a 30% glucose solution was infused at an empirically determinedrate to maintain the basal glucose level.

A bolus of 4 μCi 3-³ H-glucose solution followed by a continuousinfusion of a 10 μCi/ml solution of 3-³ H-glucose at a rate of 20 ml/min(effective dose 0.2 μCi/min) starting 120 minutes before the insulin andcold glucose infusion and continuing throughout the clamp was made.Insulin or analog infusion was started at time 0. Aliquots of wholeblood were obtained at -20, -10 and 0 minutes before the clamp wasstarted, and later at 70, 80, and 90 minutes into the clamp and theplasma glucose level determined. Glucose specific activity at thesetimes was also determined as follows: ##EQU1##

The blood was deproteinized with 10% TCA, centrifuged, and an aliquot ofsupernatant dried in a vacuum oven to evaporate off ³ H₂ O. The ³H-glucose levels in the sample were determined using a liquidscintillation counter. Basal R_(a) (glucose appearance rate) and clampR_(a) were calculated by using steady state glucose specific activity(basal: -20, -10 and ? minutes; clamp: 70, 80, 90 minutes) and thefollowing equation: ##EQU2## Under basal conditions, at steady state,R_(a) =R_(d) and R_(a) hepatic glucose output. Under clamp conditions,at a steady state, R_(a) =R_(d), however under these conditions, R_(a)=hepatic glucose output plus exogenous glucose infusion rate. Hepaticglucose output under clamp conditions was determined by subtracting theglucose infusion rate from the calculated R_(d).

In Vivo Results

A summary of the in vivo data is found in Table I.

A12Gly

A12Gly insulin analog was tested in animals at 25.5, 83.0, 166, and 255ng/kg/min. When A12gly was infused at 25.5 ng/kg/min, the R_(d) was 12.8mg/kg/min and HGO was 9.7 mg/kg/min (FIG. 3 and Table I). Thisrepresents little, if any, stimulation of R_(d) (1.03 fold) and a 22%suppression of HGO. At the 83 ng/kg/min dose, R_(d) was 14.4 mg/kg/min(1.16 fold stimulation) and HGO was 5.2 mg/kg/min (58% suppression). Atthe 166 and 255 ng/kg/min doses, R_(d) was 19.1 and 23.8 mg/kg/min,respectively, and HGO was 1.5 and 2.1 mg/kg/min, respectively. This isequivalent to a 1.5 and 1.9 fold stimulation of R_(d) and an 88% and 83%suppression of HGO for these two doses. From these data, it appears thatA12Gly is more potent than insulin at the liver and slightly less potentthan insulin at the periphery. A12Gly has approximately 1.9 the potencyof insulin in suppressing HGO and 0.75 the potency of insulin instimulating R_(d) indicating that A12Gly is hepatoselective. The ratioof in vivo liver to peripheral activity is approximately 2.6.

A19His

The analog was tested at 25.5, 76.5, 255, and 373 ng/kg/min doses.Results of the studies with A19His and insulin are shown in FIG. 4 andTable I. The glucose infusion rates necessary to maintain euglycemia atthe 25.5 and 76.5 ng/kg/min doses of A19His were very low (3.1 and 2.0mg/kg/min, respectively). Because of this result, the analog was infusedat higher doses. At the 255 ng/kg/min dose, R_(d) was 15.7 mg/kg/min(1.16 fold stimulation) and HGO was 7.4 mg/kg/min (45% suppression). Atthe 373 ng/kg/min dose, R_(d) was 16.0 mg/kg/min (1.19-fold stimulation)and HGO was 7.2 mg/kg/min (47% suppression). These results indicate thatwhen HGO is inhibited by approximately 50%, there is very littlestimulation of R_(d). By comparing the data for A19His to that forinsulin, it can be seen that the potency of A19His is less than that forinsulin. However, the potency of A19His in suppressing HGO(approximately 0.3) is greater than its potency in stimulating R_(d). Todetermine the peripheral potency (R_(d)) and the in vivo ratio ofhepatic activity to peripheral activity from these data, it wasnecessary to extrapolate the R_(d) curve. We estimate the peripheralpotency to be approximately 0.10 and the ratio to be approximately 3.These results indicate that A19His is hepatoselective in vivo.

A12Gly/A19His

A12Gly/A19His, prepared in a similar manner to the analogs describedabove, contains two substitutions in one molecule which have been shownto be hepatoselective individually. Animals were tested at 100ng/kg/min, 333 ng/kg/min, and 1000 ng/kg/min. At the 100 ng/kg/min dose,R_(d) was 14.5 mg/kg/min (1.1-fold simulation) and HGO was 8.3 mg/kg/min(36% suppression). At the 333 ng/kg/min dose, R_(d) was 16.7 mg/kg/min(1.3-fold stimulation) and HGO was 217 mg/kg/min (79% suppression). At adose of 1000 ng/kg/min, R_(d) is stimulated by 2.0-fold and HGO issuppressed by 80% (Table I). These results suggest that A12gly/A19his ishepatoselective (Table I). When compared to the animals treated withA19His, A12Gly/A19His is similar to A19His in stimulating R_(d) and ismore potent in suppressing HGO than is A19His. A12Gly/A19His appears tobe 4-fold hepatoselective.

A19G]n

A19Gln, prepared in a similar manner to the analogs described above, wastested at 333 and 1000 ng/kg/min doses (FIG. 4 and Table I). At the 333ng/kg/min dose, R_(d) was 13.3 mg/kg/min (1.1-fold stimulation) and HGOwas 7.4 mg/kg/min (40% suppression). At the 1000 ng/kg/min dose, R_(d)was 14.7 (1.2-fold stimulation) and HGO was 6.1 (51% suppression). Thein vivo data are similar to the results obtained with A19His andindicate that A19Gln is hepatoselective.

A14Phe and A10Pro/A13Trp

Each analog, prepared in a similar manner to the analogs describedabove, was infused at 100 and 333 ng/kg/min. R_(d) was stimulated toapproximately the same extent by each analog and found to be similar toinsulin stimulation (Table I). Both analogs were less effectivesuppressing HGO than was insulin indicating that they are peripheralselective.

A14Gly

A14Gly Was tested at 41.5, 100, 333, and 415 ng/kg/min. At the 41.5ng/kg/min dose, R_(d) was 13.8 mg/kg/min (1.1 fold stimulation) and HGOwas 12.3 mg/kg/min (6% suppression). At the 100 ng/kg/min dose, R_(d)was 16.3 mg/kg/min (1.4 fold stimulation) and HGO was 5.5 mg/kg/min (55%suppression). At the 333 ng/kg/min dose, R_(d) was 21.7 mg/kg/min (1.7fold stimulation) and HGO was 2.5 mg/kg/min (82% suppression). At the415 ng/kg/min dose, R_(d) was 22.2 mg/kg/min (1.6 fold stimulation) andHGO was 5.7 mg/kg/min (60% suppression) (Table I). These resultsindicate that A14Gly is hepatoselective in vivo.

A12Thr

A12Thr was tested at 100 and 333 ng/kg/min. At the 100 ng/kg/min dose,R_(d) was 20.1 ng/kg/min (1.6 fold stimulation) and HGO was 9.6mg/kg/min (24% suppression). At the 333 ng/kg/min dose, R_(d) was 39.2ng/kg/min (3.1 fold stimulation) and HGO was 3.4 mg/kg/min (69%suppression). These results indicate that A12Thr is peripheralselective.

                  TABLE I                                                         ______________________________________                                        SUMMARY OF IN VIVO DATA                                                       ______________________________________                                                    R.sub.d    HGO        GIR                                         Analog      mg/kg/min  mg/kg/min  mg/kg/min                                   ______________________________________                                        Insulin                                                                       basal       13.5 ± 0.4                                                                            13.5 ± 0.4                                                                            0                                           41.5        13.0 ± 0.3                                                                            11.3 ± 0.3                                                                            1.7 ± 0.6                                83          14.7 ± 1.3                                                                            7.8 ± 1.1                                                                             6.9 ± 0.8                                100         17.8 ± 1.0                                                                            9.1 ± 0.7                                                                             8.8 ± 1.2                                166         26.1 ± 0.8                                                                            6.4 ± 1.1                                                                             19.7 ± 0.5                               415         34.3 ± 1.6                                                                            3.5 ± 0.7                                                                             30.8 ± 1.7                               Human A12Gly                                                                  basal        12.4 ± 0.68                                                                          12.4 ± 0.68                                                                           0                                           25.5        12.8 ± 2.1                                                                            9.7 ± 2.4                                                                             3.0 ± 0.8                                83.0        14.4 ± 0.8                                                                            5.2 ± 0.4                                                                             9.2 ± 1.2                                166         19.1 ± 1.4                                                                            1.5 ± 1.1                                                                             17.7 ± 2.2                               255         23.8 ± 1.4                                                                            2.1 ± 1.4                                                                             21.7 ± 1.3                               Human A19His                                                                  basal       13.5 ± 0.8                                                                            13.5 ± 0.8                                                                            0                                           25.5        12.5        9.4       3.1                                         76.5        12.6       10.6       2.0                                         255         15.7 ± 0.6                                                                            7.4 ± 0.9                                                                             8.3 ± 1.2                                373         16.0 ± 0.7                                                                            7.2 ± 0.4                                                                             8.8 ± 0.9                                Human A19Gln                                                                  basal       12.5 ± 0.7                                                                            12.5 ± 0.7                                                                            0                                           333         13.1 ± 1.0                                                                            7.4 ± 1.1                                                                             5.8 ± 0.3                                1000        14.7 ± 0.9                                                                            6.1 ± 1.0                                                                             8.6 ± 0.6                                Human A12Gly/A19His                                                           basal       13.0 ± 0.4                                                                            13.0 ± 0.4                                                                            0                                           100         14.5 ± 0.6                                                                            8.3 ± 1.0                                                                             6.1 ± 1.0                                333         16.7 ± 1.8                                                                            1.7 ± 0.6                                                                             14.0 ± 2.0                               1000        26.4 ± 2.6                                                                            3.1 ± 1.1                                                                             23.4 ± 1.6                               Human A14Phe                                                                  basal       15.5 ± 0.6                                                                            15.5 ± 0.6                                                                            0                                           100         21.2 ± 0.9                                                                            12.9 ± 1.4                                                                            8.4 ± 2.3                                333         32.3 ± 4.0                                                                            6.2 ± 2.6                                                                             26.1 ± 2.9                               Human A10Pro/A13Trp                                                           basal       12.7 ± 1.2                                                                            12.7 ± 1.2                                                                            0                                           100         18.8 ± 1.9                                                                            10.2 ± 1.8                                                                            8.5 ± 0.5                                333         31.3 ± 1.9                                                                            8.9 ± 2.7                                                                             22.4 ± 1.9                               Human A12Ala                                                                  basal       13.6 ± 0.9                                                                            13.6 ± 0.9                                                                            0                                           100         19.3 ± 1.3                                                                            9.7 ± 1.3                                                                             9.6 ± 0.4                                333         29.1 ± 2.7                                                                            6.1 ± 0.9                                                                             22.9 ± 3.1                               1000        48.6 ± 2.1                                                                            6.2 ± 0.5                                                                             42.4 ± 2.2                               ______________________________________                                                    R.sub.d    HGO        M                                           Analog      mg/kg/min  mg/kg/min  mg/kg/min                                   ______________________________________                                        A14Gly                                                                        basal       13.2 ± 0.6                                                                            13.2 ± 0.6                                                                            0                                           41.5        13.8 ± 1.1                                                                            12.3 ± 0.8                                                                            1.6 ± 0.5                                100         16.3 ± 0.8                                                                            5.5 ± 1.2                                                                             10.8 ± 1.0                               333         21.7 ± 1.5                                                                            2.5 ± 1.1                                                                             19.5 ± 2.2                               415         22.2 ± 1.0                                                                            5.7 ± 0.9                                                                             16.5 ± 1.7                               A12Thr                                                                        basal       12.7 ± 0.4                                                                            12.7 ± 0.4                                                                            0                                           100         20.1 ± 0.8                                                                            9.6 ± 1.2                                                                             10.5 ± 0.6                               333         39.2 ± 1.5                                                                            3.4 ± 1.1                                                                             35.9 ± 1.6                               ______________________________________                                    

The instant invention as shown and described herein was considered to bethe most practical and preferred embodiment. It is recognized, however,that departures may be made therefrom which are within the scope of theinvention, and that obvious modifications will occur to one skilled inthe art upon reading this disclosure.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCGG GGTTCTATGTTTGTGAACCAACACCTGTGCGGATCCCACCTGGTGGAAGCTCTCTA60               CCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCC97                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 89 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAAGCCTCGTTCCCCGCACACTAGGTAGAGAGCTTCCACCAGGTGGGATCCGCACAGGTG60                TTGGTTCACAAACATAGAACCCCGGGTAC 89                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTG GGCGGGGGCCCT60               GGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCC99                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGCCAAGGGCTGCAGGCTGCCTGCACCAGGGCCCCCGCCCAGCTCCACCTGCCCCACCTG60                CAGGTCCTCTGCCTCCCGGCGGGTCTTGGGTGTGTAGAA99                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 79 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGTATCTGCTCCCTCTACCAGCTG60                GAGAACTACTGCAACTAGA 79                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGCTTCTAGTTGCAGTAGTTCT CCAGCTGGTAGAGGGAGCAGATACTGGTACAGCATTGT60               TCCACAATGCCACGCTTCTGCAGGGACCCCTCCAG95                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 510 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..510                                                          (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGG48                            MetGluLys LysIleThrGlyTyrThrThrValAspIleSerGlnTrp                             151015                                                                        CATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACC96                            Hi sArgLysGluHisPheGluAlaPheGlnSerValAlaGlnCysThr                             202530                                                                        TATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTA144                            TyrAsnGlnThrValGlnLeuAspIleThrAlaPheLeuLysThrVal                             354045                                                                        AAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCC192                            LysLysAsnLysHisLysPheTyrProAlaPheIleHisIleLeuAla                             505560                                                                        CGCCTGATGAATGCTCATCCGGAATTCGAGCTCGGTACCCGGGGTTCT240                            ArgLeuMetAsnAlaHisProGluPheGluLeuGlyThrArgGlySer                             65707580                                                                      ATGTTTGTGAACCAACACCTGTGTGGATCCCACCTGGTGGAAGCTC TC288                          MetPheValAsnGlnHisLeuCysGlySerHisLeuValGluAlaLeu                              859095                                                                        TACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCC AAGACCCGC336                          TyrLeuValCysGlyGluArgGlyPhePheTyrThrProLysThrArg                              100105110                                                                     CGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAG CTGGGCGGGGGC384                          ArgGluAlaGluAspLeuGlnValGlyGlnValGluLeuGlyGlyGly                              115120125                                                                     CCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGA GGGGTCCCTGCAG432                          ProGlyAlaGlySerLeuGlnProLeuAlaLeuGluGlySerLeuGln                              130135140                                                                     AAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCT GCTCCCTCTAC480                          LysArgGlyIleValGluGlnCysCysThrSerIleCysSerLeuTyr                              145150155160                                                                  CAGCTGGAGAACTACTGCAACTAGAAG CTT510                                            GlnLeuGluAsnTyrCysAsn                                                         165167                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 167 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (x i) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      MetGluLysLysIleThrGlyTyrThrThrValAspIleSerGlnTrp                              151015                                                                        HisArgLysGluHisPheGluAlaPheGlnSerValAla GlnCysThr                             202530                                                                        TyrAsnGlnThrValGlnLeuAspIleThrAlaPheLeuLysThrVal                              354045                                                                        LysLysAsnLysHisLysPheTyrProAlaPheIleHisIleLeuAla                              505560                                                                        ArgLeuMetAsnAlaHisProGluPheGluLeuGlyThrArgGlySer                               65707580                                                                     MetPheValAsnGlnHisLeuCysGlySerHisLeuValGluAlaLeu                              859095                                                                         TyrLeuValCysGlyGluArgGlyPhePheTyrThrProLysThrArg                             100105110                                                                     ArgGluAlaGluAspLeuGlnValGlyGlnValGluLeuGlyGlyGly                               115120125                                                                    ProGlyAlaGlySerLeuGlnProLeuAlaLeuGluGlySerLeuGln                              130135140                                                                     LysArgGlyIle ValGluGlnCysCysThrSerIleCysSerLeuTyr                             145150155160                                                                  GlnLeuGluAsnTyrCysAsn                                                         165167                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCGGAATTCGAGCTCGGTACCCGG24                                                    (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCCAAGCTTCTAGTTGCAGTAGTTCTCCAGCTGGTAGAGACCGCA45                               (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCCAAGCTTCTAGTTGCAATGGTTTTC27                                                 (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 275 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 209..271                                                        (D) OTHER INFORMATION: /note="The insulin analog                              is numbered from amino acid number 1 of the                                    A chain."                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCGGGGTTCTATGTTTGTGAACCAACACCTGTGCGGATCCCACCTGGTGGAAGCTCTCTA60                CCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGA120               CCTGCAGGTGGGGCAG GTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTT180              GGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACC232                       GlyIleValGluGlnCysCysThr                                                       15                                                                           AGTATCTGCGGTCTCTACCAGCTGGAGAACTACTGCAACTAGA275                                SerIleCysGlyLeuTyrGlnLeuGluAsnTyrCysAsn                                       10 1520                                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GlyIleValGluGlnCysCysThrSerIleCysGly LeuTyrGlnLeu                             151015                                                                        GluAsnTyrCysAsn                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 275 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 209..271                                                        (D) OTHER INFORMATION: /note="The insulin analog                              is numbered from amino acid number 1 of the                                   A chain."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCGGGGTTCTATGTTTGTGAACCA ACACCTGTGCGGATCCCACCTGGTGGAAGCTCTCTA60               CCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGA120               CCTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTT180               G GCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACC232                      GlyIleValGluGlnCysCysThr                                                      15                                                                            A GTATCTGCTCCCTCTACCAGCTGGAGAACCATTGCAACTAGA275                               SerIleCysSerLeuTyrGlnLeuGluAsnHisCysAsn                                       101520                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyIleValGluGlnCysCysThrSerIleCysSerLeuTyrGlnLeu                              151 015                                                                       GluAsnHisCysAsn                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 276 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                              (B) LOCATION: 11..272                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CCGGGGTTCTATGTTTGTGAACCAACACCTGTGCGGATCCCACCTGGTG49                           MetPheValAsnGlnHisLeuCysGlySerHisLeuVal                                       15 10                                                                         GAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCC97                            GluAlaLeuTyrLeuValCysGlyGluArgGlyPhePheTyrThrPro                              1520 25                                                                       AAGACCCTGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCT145                           LysThrLeuProGlyGlyArgGlyProAlaGlyGlyAlaGlyGlyAla                              303540 45                                                                     GGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGG193                           GlyArgGlyProTrpCysArgGlnProAlaAlaLeuGlyProGlyGly                              5055 60                                                                       GTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGTATCTG241                           ValProAlaGluAlaTrpHisCysGlyThrMetLeuTyrGlnTyrLeu                              6570 75                                                                       CTCCCTCTACCAGCTGGAGAACTACTGCAACTAGA276                                        LeuProLeuProAlaGlyGluLeuLeuGln                                                8085                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      MetPheValAsnGlnHisLeuCysGlySerHisLeuValGluAlaLeu                              151015                                                                        Ty rLeuValCysGlyGluArgGlyPhePheTyrThrProLysThrLeu                             202530                                                                        ProGlyGlyArgGlyProAlaGlyGlyAlaGlyGlyAlaGlyArgGly                              35 4045                                                                       ProTrpCysArgGlnProAlaAlaLeuGlyProGlyGlyValProAla                              505560                                                                        GluAlaTrpHisCysGlyThrMetLeuTyrGlnT yrLeuLeuProLeu                             65707580                                                                      ProAlaGlyGluLeuLeuGln                                                         85                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 276 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AGATCAACGTCATCAAGAGGTCGACCATCTCCCTCGTCTATGACCATGTCGTAACAAGGT60                GTTACGGTGCGAAGACGTCCCTGGGGAGGTCCCGGTTCCCGACGTCCGACGGACGTGGTC120               C CGGGGGCGGGTCGAGGTGGACGGGGTGGACGTCCAGGAGACGGAGGGCCGTCCCAGAAC180              CCACACATCTTCTTCGGAGCAAGGGGCGTGTGATCCATCTCTCGAAGGTGGTCCACCCTA240               GGCGTGTCCACAACCAAGTGTTTGTATCTTGGGGCC 276                                  

We claim:
 1. A human insulin analog wherein position A12_(Ser) issubstituted with Gly and the analog is hepatoselective.
 2. A humaninsulin analog wherein position A12_(Ser) is substituted with Thr andthe analog is peripherally selective.
 3. A human insulin analogcomprising a hydrophilic amino acid substitution at position A19_(Tyr),where the analog is hepatoselective.
 4. The insulin analog of claim 3,wherein position A19_(Tyr) is substituted by either His or Gln.
 5. Ahuman insulin analog comprising amino acid substitutions at bothpositions A12_(Ser) and A19_(Tyr), wherein position A12_(Ser) issubstituted with Gly and A19_(Tyr) is substituted with His and theanalog is hepatoselective.
 6. A human insulin analog comprising ahydrophobic amino acid substitution at position A14_(Tyr), wherein theanalog is peripherally selective.
 7. The insulation analog of claim 6,wherein position A14_(Tyr) is substituted with Phe.
 8. A human insulinanalog comprising a hydrophilic amino acid substitution at positionA14_(Tyr), wherein the analog is hepatoselective.
 9. The insulin analogof claim 8, wherein position A14_(Tyr) is substituted with Gly.
 10. Ahuman insulin analog comprising amino acid substitutions at bothpositions A12_(Ser) and A14_(Tyr), wherein position A12_(Ser) issubstituted with Thr and position A14_(Tyr) is substituted with Gly andthe analog is hepatoselective.
 11. A human insulin analog comprisinghydrophobic amino acid substitutions at both positions A10_(Val) andA13_(Leu), wherein the analog is peripherally selective.
 12. The insulinanalog of claim 11, wherein position A10_(Val) is substituted with Proand position A13_(Leu) is substituted with Trp.